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The purpose of this study was to identify critical technical points that lead to increased ball speed in a maximal toe kick with no run-up (a 'static kick') in blind football. Six visually impaired male players and eight sighted male players participated in the experiment. All participants wore a blindfold to fully remove visual information and performed the static kick. The motion was captured three-dimensionally using an optical motion analysis system. Our results demonstrated that ball speed, maximum linear velocity of the kicking-side thigh, and maximum angular velocity of the kicking-side shank for the sighted player group were significantly greater than those for the visually impaired player group. The sighted players tended to perform the static kick in a similar motion pattern, which was characterised by a backwards rotation of the torso to adequately extend the kicking-side hip joint during the back-swing phase and a stable posture of the lower torso on the frontal plane during the forward-swing phase. This motion pattern is critical to both acceleration of the kicking-side foot and orientation of the foot for a more precise ball contact position.

〔目的〕運動前のタウリン摂取が，運動によって生じる筋疲労に与える影響を明らかにすることとした．〔対象〕大学男子サッカー選手10名とした．〔方法〕無作為化二重盲検クロスオーバー試験にて，タウリン水，プラセボ水の2種類を摂取し，75％VO2maxでのエルゴメータによる運動と，最大努力での等速性膝伸展運動を100回行った．測定項目は，血液成分，膝伸展運動中のピークトルク，および大腿直筋の平均周波数（MPF）とした．〔結果〕タウリン水摂取群は血中MB濃度の上昇と，MPFの低下が抑制される傾向を示した．〔結語〕タウリン水摂取が運動によって生じる筋損傷を抑制する可能性があることが示唆される．, [Purpose] The purpose of this study was to investigate the effect of taurine ingestion before exercise on muscle fatigue. [Subjects] Ten male soccer players participated in this study. [Methods] This study had a double-blind crossover test design. The subjects drank taurine water or placebo water before exercise. Two types of exercise were performed: bicycle ergometer and isokinetic knee extension exercise. Muscle fatigue was evaluated using the mean power frequency (MPF) of the rectus femoris electromyogram, peak torque, and blood components. [Results] The taurine blood level of the subjects who imbibed taurine water was significantly higher than that of the subjects who drank placebo water. Furthermore, their myoglobin blood level was lower and their MPF showed a tendency to decline less than that of the placebo group. [Conclusion] These results suggest that the taurine blood level is increased by taurine intake, and that muscle fatigue due to exercise was inhibited by taurine ingestion.

BACKGROUND AND OBJECTIVE: Sitting posture may often place large burden on trunk muscles, while trunk muscle activities in the sitting posture have not been well clarified. In this study, a difference in trunk muscle activity between two kinds of sitting postures was evaluated, focusing on low back pain induced by posture holding. MATERIAL AND METHODS: An experiment was conducted on the subjects sitting on a stable-seat and on an unstable-seat, with the pelvis inclined forward, backward, rightward, and leftward. RESULTS: With the pelvis inclined forward, rightward and leftward, muscle activities were significantly increased in a stable-seat sitting posture. In contrast, no significant increase in muscle activity was observed with the pelvis inclined in every direction in an unstable-seat sitting posture. CONCLUSIONS: With the pelvis inclined in the stable-seat sitting posture, muscle activities were imbalanced, while with the pelvis inclined in the unstable-seat sitting posture, muscle activities were not imbalanced. Thus, it is suggested that with the pelvis inclined to the maximum extent in the stable-seat sitting posture, low back pain may be induced by imbalanced muscle activities.

Class V myosins play a pivotal role in organelle distribution. In the budding yeast, Myo2p, a class V myosin, is essential for mitochondrial distribution. We identified MMR1 as a high-dose suppressor of the myo2 mitochondrial defect and that Mmr1p resides restrictively on the bud-localizing mitochondria and forms a complex with Myo2p tail. Mmr1p loss delayed mitochondrial transfer to buds and completely abolished mitochondrial distribution in the absence of Ypt11p, which promotes mitochondrial distribution by complex formation with Myo2p tail. The myo2-573 mutation, which causes a mitochondrial distribution defect and inactivates the Mmr1p function, reduced association between Myo2p and Mmr1p and depolarized Mmr1p localization on mitochondria. These strongly suggest that Mmr1p is a key mitochondrial component of the link between Myo2p and mitochondria for Myo2p-dependent mitochondrial distribution. Genetical analysis revealed that the Mmr1p–Myo2p pathway is independent of the Ypt11p–Myo2p pathway, suggesting that an essential system for mitochondrial distribution is composed of two independent Myo2p pathways.

We identified a novel Rho gene rho3+ and studied its interaction with diaphanous/formin for3+ in the fission yeast Schizosaccharomyces pombe. Both rho3 null cells and for3 null cells showed defects in organization of not only actin cytoskeleton but also cytoplasmic microtubules (MTs). rho3 for3double null cells had defects that were more severe than each single null cell: polarized growth was deficient in the double null cells. Function of For3 needed the highly conserved FH1 and FH2 domains, an N-terminal region containing a Rho-binding domain, and the C-terminal region. For3 bound to active forms of both Rho3 and Cdc42 but not to that of Rho1. For3 was localized as dots to the ends of interphase cells and to the mid-region in dividing cells. This localization was probably dependent on its interaction with Rho proteins. Overexpression of For3 produced huge swollen cells containing depolarized F-actin patches and thick cytoplasmic MT bundles. In addition, overexpression of a constitutively active Rho3Q71L induced a strong defect in cytokinesis. In conclusion, we propose that the Rho3-For3 signaling system functions in the polarized cell growth of fission yeast by controlling both actin cytoskeleton and MTs.

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p is essential for growth and is mainly localized in the nucleolus. The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly. Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA.

A secretory defect leads to transcriptional repression of both ribosomal protein and rRNA genes in yeast. To elucidate the mechanism of the signaling, we previously isolated rrs mutants that were unable to respond to a secretory defect, and we cloned RRS1 encoding a nuclear protein that was required for ribosome biogenesis (Tsuno, A., Miyoshi, K., Tsujii, R., Miyakawa, T., and Mizuta, K. (2000) Mol. Cell. Biol. 20, 2066-2074). We identified duplicated genes encoding ribosomal protein L11, RPL11B as a wild-type allele complementing the rrs2 mutation, and RPL11A in two-hybrid screening using RRS1 as bait. Rpl11p was copurified with Rrs1p in immunoprecipitation analysis. Ultracentrifugation analysis revealed that Rrs1p associated fairly tightly with 60 S preribosomal subunits. These results suggest that signaling in response to a secretory defect requires the normal assembly of 60 S ribosomal subunits including Rrs1p and Rpl11p.

Unconventional myosins in the budding yeast play essential roles in diverse cellular functions, inacluding endocytosis, actin organization, and polarized distribution of organelles. Several lines of evidence suggest that novel proteins, interacting with the unconventional myosins, regulate their functions. In this review, we focus on the functions of unconventional myosins from the point of view of myosin-interacting proteins.

Background A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response. Results EBP2, encoding the yeast homologue of a human protein that interacts with Epstein–Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait. The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells. The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes. Conclusions Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis.

Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a small GTPase that maintains cell polarity. Sro7p and Sro77p, a homologue of Sro7p, possess domains homologous to the protein that are encoded by the Drosophila tumor suppressor gene lethal (2) giant larvae [l(2)gl]. sro7Δ sro77Δ mutants showed a partial defect of organization of the polarized actin cytoskeleton and a cold-sensitive growth phenotype. A human counterpart of l(2)gl could suppress the sro7Δ sro77Δ defect. Similar to the l(2)gl protein, Sro7p formed a complex with Myo1p, a type II myosin. These results indicate that Sro7p and Sro77p are the yeast counterparts of the l(2)gl protein. Our genetic analysis revealed that deletion of SRO7 and SRO77 showed reciprocal suppression with deletion of MYO1 (i.e., the sro7Δ sro77Δ defect was suppressed by myo1Δ and vice versa). In addition, SRO7 showed genetic interactions with MYO2, encoding an essential type V myosin: Overexpression of SRO7 suppressed a defect in MYO2 and, conversely, overexpression of MYO2 suppressed the cold-sensitive phenotype of sro7Δ sro77Δ mutants. These results indicate that Sro7 function is closely related to both Myo1p and Myo2p. We propose a model in which Sro7 function is involved in the targeting of the myosin proteins to their intrinsic pathways.

The fission yeast plc1 + gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1 + as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method. As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells. In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1 + and rad24 + belong to the same epistasis group with respect to UV sensitivity. Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plc1 null mutant cells in our analysis. Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins.

RHO3 encodes a Rho-type small GTPase in the yeast Saccharomyces cerevisiae and is involved in the proper organization of the actin cytoskeleton required for bud growth. SRO9 (YCL37c) was isolated as a multicopy suppressor of a rho3delta mutation. An Sro9p domain required for function is similar to a domain in the La protein (an RNA-binding protein). Disruption of SRO9 did not affect vegetative growth, even with the simultaneous disruption of an SRO9 homologue, SRO99. However, sro9delta was synthetically lethal with a disruption of TPM1, which encodes tropomyosin; sro9delta tpm1delta cells did not distribute cortical actin patches properly and lysed. We isolated TPM2, the other gene for tropomyosin, as a multicopy suppressor of a tpm1delta sro9delta double mutant. Genetic analysis suggests that TPM2 is functionally related to TPM1 and that tropomyosin is important but not essential for cell growth. Overexpression of SRO9 suppressed the growth defect in tpm1delta tpm2delta cells, disappearance of cables of actin filaments in both rho3delta cells and tpm1delta cells, and temperature sensitivity of actin mutant cells (act1-1 cells), suggesting that Sro9p has a function that overlaps or is related to tropomyosin function. Unlike tropomyosin, Sro9p does not colocalize with actin cables but is diffusely cytoplasmic. These results suggest that Sro9p is a new cytoplasmic factor involved in the organization of actin filaments.

Genes belonging to the ras superfamily encode low-molecular-weight GTP/GDP-binding proteins that are highly conserved in wide variety of organisms. We used the polymerase chain reaction (PCR) to isolate a novel member of the ras superfamily from the filamentous fungus Neurospora crassa and obtained a mammalian Krev-1 homolog. We named the gene krev-1 and analyzed its structure and function. The krev-1 gene encodes a polypeptide of 225 amino acids, which is nearly 60% homologous to the mammalian Krev-1 p21. The krev-1 gene product (KREV1) is functionally analogous to mammalian Krev-1 p21 and Rsr1p/Bud1p, a Krev-1 homolog from the yeast Saccharomyces cerevisiae. GAL1-driven expression of KREV1 in a wild-type yeast strain resulted in a random budding pattern, as did its mammalian counterpart Krev-1 p21. We disrupted the krev-1 gene by RIP (repeat-induced point mutation), but the krev-1 disruptants showed no abnormalities. By in vitro mutagenesis, we constructed several mutant krev-1 genes (G21V, A68T, and D128A) which mimic constitutively active mutants of Ha-ras, and the krev-1 (K25N) mutant which is analogous to a dominant-negative mutant of Ha-ras. Each mutant gene was introduced into the wild-type strain and the phenotypes were analyzed. We could not observe any difference in vegetative growth between these transformants. When each strain was used as the female in mating tests, the development of perithecia from protoperithecia was inhibited in all cases. The results indicate that the krev-1 gene may be involved in sexual cycle progression.

The yeast protein Bem1p, which bears two src homology region 3 (SH3) domains, is involved in cell polarization. A Rho-type GTPase, Rho3p, is involved in the maintenance of cell polarity for bud formation, and the rho3 defect is suppressed by a high dose of BEM1. Mutational analysis revealed that the second SH3 domain from the NH2 terminus (SH3-2) of Bem1p is important for the functions of Bem1p in bud formation and in the suppression of the rho3 defect. Boi2p, which bound to SH3-2 Bem1p, was identified using the two-hybrid system. Boi2p has a proline-rich sequence that is critical for displaying the Boi2p-Bem1p two-hybrid interaction, an SH3 domain in its NH2-terminal half, and a pleckstrin homology domain in its COOH-terminal half. A BOI2 homologue, BOI1, was identified as a gene whose overexpression inhibited cell growth. Cells overexpressing either BOI1 or BOI2 were arrested as large, round, and unbudded cells, indicating that the Boi proteins affect cell polarization. Genetic analysis revealed that BOI1 and BOI2 are functionally redundant and important for cell growth. delta boi1 delta boi2 cells became large round cells or lysed with buds, displaying defects in bud formation and in the maintenance of cell polarity. Analysis using several truncated versions of BOI2 revealed that the COOH-terminal half, which contains the pleckstrin homology domain is essential for the function of Boi2p in cell growth, while the NH2-terminal half is not, and the NH2-terminal half might be required for modulating the function of Bem1p. Overproduction of either Rho3p or the Rho3p-related GTPase Rho4p suppressed the boi defect. These results demonstrate that Rho3p GTPases and Boi proteins function in the maintenance of cell polarity for bud formation.

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase in Saccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity of lte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that of lte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic to CDC15, which encodes a protein kinase. The cdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression of lte1. We isolated CDC14 as a low-copy-number suppressor of cdc15-rlt1. CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy of CDC14 suppressed the temperature sensitivity of cdc15-rlt1 cells, but not that of cdc15-1 cells. In addition, some residues that are essential for the CDC14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.

The PLC1 gene of the yeast Saccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitive plc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutant plc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature, plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests that PLC1 is required at several or all stages of the cell cycle.

LTE1 belongs to the CDC25 family that encodes a guanine nucleotide exchange factor for GTP-binding proteins of the ras family. Previously we have shown that LTE1 is essential for termination of M phase at low temperatures. We have identified TEM1 as a gene that, when present on a multicopy plasmid, suppresses the cold-sensitive phenotype of lte1. Sequence analysis of TEM1 and GTP-binding analysis of the gene product revealed that TEM1 encodes a novel low-molecular-weight GTP-binding protein. The defect of TEM1 was lethal, and the tem1-defective cells were arrested at telophase with high H1-kinase activity under restrictive conditions, indicating that TEM1 is required to exit from M phase. The defect of TEM1 was suppressed by a high dose of CDC15, which encodes a protein kinase homologous to mitogen-activated protein kinase kinase kinases. The genetic interaction among LTE1, TEM1, and CDC15 indicates that they cooperatively play an essential role for termination of M phase.

We have identified MSI2 as a gene of Saccharomyces cerevisiae which, when on a multicopy vector, suppresses the heat shock sensitivity caused by the loss of the IRA1 product, a negative regulator of the RAS protein. The multicopy MSI2 also suppresses the heat shock sensitivity of cells with the RAS2val19 mutation but not those with the bcy1 mutation, suggesting that the MSI2 protein may interfere with the activity of the RAS protein. The sequence analysis of MSI2 reveals that it is identical to LTE1 belonging to the CDC25 family: CDC25, SCD25 and BUD5, each of which encodes a guanine nucleotide exchange factor for the ras superfamily gene products. Deletion of the entire MSI2 coding region reveals that MSI2 is not essential but the disruptant shows a cold-sensitive phenotype. Under the non-permissive conditions, more than 70% of the msi2 disruptants arrested at telophase as large budded cells with two nuclei divided completely and elongated spindles, indicating that the msi2 deletion is a cell division cycle mutation. These results suggest that MSI2 is involved in the termination of M phase and that this process is regulated by a ras superfamily gene product.

We have examined expression of the N-myc, c-fos and smg p25A genes in two human neuroblastoma cell lines during their differentiation. The decrease in the N-myc gene expression and the increase in the c-fos gene expression are observed during the differentiation of NB-1 cells into neuronal cells and of GOTO cells into Schwann-type cells. On the other hand, the smg p25A, a ras p21-like small GTP-binding protein, gene expression is increased in NB-1 cells but not in GOTO cells during their differentiation, suggesting that smg p25A is closely associated with the neuronal phenotype of neuroblastoma cells.

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.

The budding yeast Saccharomyces cerevisiae has three classes of myosins corresponding to three actin structures: class I myosin for endocytic actin structure, actin patches; class II myosin for contraction of the actomyosin contractile ring around the bud neck; and class V myosin for transport along a cable-like actin structure (actin cables), extending toward the growing cortex. Myo2p and Myo4p constitute respective class V myosins as the heavy chain and, like class V myosins in other organisms, function as actin-based motors for polarized distribution of organelles and intracellular molecules. Proper distribution of organelles is essential for autonomously replicating organelles that cannot be reproduced de novo, and is also quite important for other organelles to ensure their efficient segregation and proper positioning, even though they can be newly synthesized, such as those derived from endoplasmic reticulum. In the budding yeast, microtubule-based motors play limited roles in the distribution. Instead, the actin-based motor myosins, especially Myo2p, play a major role. Studies on Myo2p have revealed a wide variety of Myo2p cargo and Myo2p-interacting proteins and have established that Myo2p interacts with cargo and transfers it along actin cables. Moreover, recent findings suggest that Myo2p has another way to distribute cargo in that Myo2p conveys the attaching cargo along the actin track. Thus, the myosin have "dual paths" for distribution of a cargo. This dual path mechanism is proposed in the last section of this review.

The budding yeast Saccharomyces cerevisiae has three classes of myosins corresponding to three actin structures: class I myosin for endocytic actin structure, actin patches; class II myosin for contraction of the actomyosin contractile ring around the bud neck; and class V myosin for transport along a cable-like actin structure (actin cables), extending toward the growing cortex. Myo2p and Myo4p constitute respective class V myosins as the heavy chain and, like class V myosins in other organisms, function as actin-based motors for polarized distribution of organelles and intracellular molecules. Proper distribution of organelles is essential for autonomously replicating organelles that cannot be reproduced de novo, and is also quite important for other organelles to ensure their efficient segregation and proper positioning, even though they can be newly synthesized, such as those derived from endoplasmic reticulum. In the budding yeast, microtubule-based motors play limited roles in the distribution. Instead, the actin-based motor myosins, especially Myo2p, play a major role. Studies on Myo2p have revealed a wide variety of Myo2p cargo and Myo2p-interacting proteins and have established that Myo2p interacts with cargo and transfers it along actin cables. Moreover, recent findings suggest that Myo2p has another way to distribute cargo in that Myo2p conveys the attaching cargo along the actin track. Thus, the myosin have "dual paths" for distribution of a cargo. This dual path mechanism is proposed in the last section of this review.

The authors propose a novel stacked capacitor cell with dual cell plate (DCP cell) for 64-Mb DRAMs. The major advantage of this cell is that the dual cell plates completely surround the whole surface of the storage polysilicon, and the storage capacitance of this cell increases significantly compared to the conventional stacked capacitor cell. For a 1.3- mu m/sup 2/ cell, the DCP cell should achieve a storage capacitance of more than 25 fF. The experimental results indicate that the DCP cell can realize the 64-Mb DRAMs and 1.3- mu m/sup 2/ cell area using the 0.3- mu m design rule. >

We identified Ypt11p, a rab-type small GTPase, by its functional and two-hybrid interaction with Myo2p, a class V myosin of the budding yeast Saccharomyces cerevisiae. The tail domain of Myo2p was coimmunoprecipitated with Ypt11p, suggesting that Ypt11p forms a complex with Myo2p at its tail domain in vivo. Mutational analysis of YPT11 suggests that Myo2p is a putative effector of Ypt11p. Deletion of YPT11 induced partial delay of mitochondrial transmission to the bud, and overexpression of YPT11 resulted in mitochondrial accumulation in the bud, indicating that Ypt11p acts positively on mitochondrial distribution toward the bud. We isolated two myo2 mutants, myo2-338 and myo2-573, which showed genetic interactions with YPT11. The myo2-573 mutation, identified by a synthetic lethal interaction with ypt11-null, induced a defect in mitochondrial distribution toward the bud, indicating that Myo2p plays a crucial role in polarized distribution of mitochondria. The myo2-338 mutation was identified as the mutation that abolished the effect of overexpressed YPT11, such as the Ypt11p-dependent accumulation of mitochondria in the bud, and the affinity of Myo2p for Ypt11p was reduced. These results indicate that complex formation of Ypt11p with Myo2p accelerates the function of Myo2p for mitochondrial distribution toward the bud.

Modular domains mediating specific protein-protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67(phox), which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40(phox) (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67(phox), which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40(phox), which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.

The room‐temperature coercivity of acicular γ‐Fe2O3 particles having cobalt ions adsorbed in the pH range from 5 to 13.5 and in the temperature range from 283 to 373 K was measured. Emission Mossbauer spectroscopy at room temperature was used to study cobalt adsorbed on the surface of acicular γ‐Fe2O3 particles. The coercivity of cobalt‐adsorbed γ‐Fe2O3 particles increased with increasing cobalt content up to about 2 wt %, above which it leveled off. The saturated values of coercivity increased with increasing adsorption temperature from 283 to 303 K, decreased with further increasing temperature up to 343 K, and again increased remarkably at 373 K. It was found from Mossbauer spectroscopy that the sample with high coercivity shows a strong magnetic interaction between the absorbed cobalt ions and the ordered magnetic ions of the substrate. Nonmagnetic components that do not contribute to the coercivity are probably due to the oxides and hydroxides of divalent and trivalent cobalts. The effect of pretreatment with Mg2+, Ca2+, and Sr2+ ions on the adsorption of cobalt on the surface of γ‐Fe2O3 at 373 K was also studied by emission Mossbauer spectroscopy. It was found that the average internal magnetic field of γ‐Fe2O3 with both Mg2+ and Co2+ ions adsorbed was larger than that for Co2+ ions alone, whereas in the case of Ca2+ and Sr2+ ions, the average internal magnetic field did not change. These phenomena are discussed on the basis of the site preference of cations in ferrites.

The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

Exploiting the polymerase chain reaction, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) of the fission yeast Schizosaccharomyces pombe. Inspection of the nucleotide sequence of the gene revealed an open reading frame that can encode a polypeptide of 899 amino acid residues with a calculated molecular mass of 102 kDa. This putative polypeptide contains both the X and Y regions that are conserved among three classes of mammalian PLC, and also contains a presumptive Ca2+-binding site (an E-F hand motif). The structure of the putative protein is most similar to that of the δ class of PLC isozymes. To investigate the role of this gene, designated plc1+, gene disruption was carried out by interrupting the coding region with the ura4+ marker. Growth of plc1 cells was temperature-sensitive in rich medium, and cells could not grow in synthetic medium. Expression of the PLC1 gene of Saccharomyces cerevisiae suppressed the growth defect phenotype of plc1− cells, a strong suggestion that the plc1+ gene encodes PLC. The PLC1 sequence appears in the public data libraries, DDBJ GenBank, EMBL under the following Accession Number: D38309.

Background Muscle contraction during short intervals of intense exercise causes oxidative stress, which can play a role in the development of overtraining symptoms, including increased fatigue, resulting in muscle microinjury or inflammation. Recently it has been said that hydrogen can function as antioxidant, so we investigated the effect of hydrogen-rich water (HW) on oxidative stress and muscle fatigue in response to acute exercise. Methods Ten male soccer players aged 20.9 ± 1.3 years old were subjected to exercise tests and blood sampling. Each subject was examined twice in a crossover double-blind manner; they were given either HW or placebo water (PW) for one week intervals. Subjects were requested to use a cycle ergometer at a 75 % maximal oxygen uptake (VO2) for 30 min, followed by measurement of peak torque and muscle activity throughout 100 repetitions of maximal isokinetic knee extension. Oxidative stress markers and creatine kinase in the peripheral blood were sequentially measured. Results Although acute exercise resulted in an increase in blood lactate levels in the subjects given PW, oral intake of HW prevented an elevation of blood lactate during heavy exercise. Peak torque of PW significantly decreased during maximal isokinetic knee extension, suggesting muscle fatigue, but peak torque of HW didn’t decrease at early phase. There was no significant change in blood oxidative injury markers (d-ROMs and BAP) or creatine kinease after exercise. Conclusion Adequate hydration with hydrogen-rich water pre-exercise reduced blood lactate levels and improved exercise-induced decline of muscle function. Although further studies to elucidate the exact mechanisms and the benefits are needed to be confirmed in larger series of studies, these preliminary results may suggest that HW may be suitable hydration for athletes.

The MSI3 gene was isolated as a multicopy suppressor of the heat shock-sensitive phenotype of the iral mutation, which causes hyperactivation of the RAS-cAMP pathway. Overexpression of MSI3 also suppresses the heat shock-sensitive phenotype of the bcyl mutant. Determination of the DNA sequence of MSI3 revealed that MSI3 can encode a 77.4 kDa protein related to the HSP70 family. The amino acid sequence of Msi3p is about 30% identical to that of the Ssalp of Saccharomyces cerevisiae. This contrasts with the finding that members of the HSP70 family generally show at least 50% amino acid identity. The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of MSI3. Moreover, the steady-state levels of the MSI3 mRNA and protein were increased upon heat shock. These results indicate that the MSI3 gene encodes a novel HSP70-like heat shock protein. Disruption of the MSI3 gene was associated with a temperature sensitive growth phenotype but unexpectedly, thermotolerance was enhanced in the disruptant.

We isolated three highly homologous genes, PIR1, PIR2 and PIR3, collectively called the PIR genes. The remarkable feature of their putative amino acid sequence is that they contain a sequence consisting of 18-19 amino acid residues repeated tandemly seven to ten times. Genes homologous to PIR were found in Kluyveromyces lactis and Zygosaccharomyces rouxii but not in Schizosaccharomyces pombe, suggesting that a set of PIR genes plays some role in budding yeast. Bias of codon usage seen in each of the PIR translation products suggests that they are expressed abundantly. The fact that disruption of each gene is viable indicates that none of them is essential. The double disruptants, pir1 pir2, were viable under various conditions, such as higher temperature (37 degrees C) or high salt concentration, but showed a slow-growing phenotype on an agar slab. Furthermore, they were sensitive to heat shock. Addition of a pir3 disruption to the pir1 pir2 double disruptant brought about no phenotypic difference from the original double mutant. PIR1 and PIR3 are closely linked to each other and are on chromosome XI.

RHO3 and RHO4 are members of the ras superfamily genes of the yeast Saccharomyces cerevisiae and are related functionally to each other. Experiments using a conditionally expressed allele of RHO4 revealed that depletion of both the RHO3 and RHO4 gene products resulted in lysis of cells with a small bud, which could be prevented by the presence of osmotic stabilizing agents in the medium. rho3 rho4 cells incubated in medium containing an osmotic stabilizing agent were rounded and enlarged and displayed delocalized deposition of chitin and delocalization of actin patches, indicating that these cells lost cell polarity. Nine genes whose overexpression could suppress the defect of the RHO3 function were isolated (SRO genes). Two of them were identical with CDC42 and BEM1, bud site assembly genes involved in the process of bud emergence. A high dose of CDC42 complemented the rho3 defect, whereas overexpression of RHO3 had an inhibitory effect on the growth of mutants defective in the CDC24-CDC42 pathway. These results, along with comparison of cell morphology between rho3 rho4 cells and cdc24 (or cdc42) mutant cells kept under the restrictive conditions, strongly suggest that the functions of RHO3 and RHO4 are required after initiation of bud formation to maintain cell polarity during maturation of daughter cells.

We have isolated two novel genes, designated as RHO3 (on chromosome IX) and RHO4 (on chromosome XI), belonging to the ras superfamily, from the yeast Saccharomyces cerevisiae . Deletion of RHO4 did not affect cell growth, but deletion of RHO3 caused a severe growth delay and a decrease in cell viability. The rho3 and rho4 disrupted genes displayed synthetic lethality at 30°C. RHO3 and RHO4 seem to have overlapping functions in cell growth, because the growth defect in rho3 cells was suppressed by overexpression of RHO4 despite the low homology (about 35%) at the amino acid sequence level between the RHO3 and RHO4 proteins.

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a brobe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev −1 proteins and smg p21B is identical with the rap1B protein.

Interface states in the ferroelectric-semiconductor junction have been investigated from analyses of DLTS andC-V data. Two trap levels are located at 0.21 and 0.36 eV below the conduction band near the silicon side of the interface in the MFS (Metal-Ferroelectric-Semiconductor) structure. The interface states density has been drastically reduced by putting an oxide layer between ferroelectric and semiconductor with certain heat treatment in H2 atmosphere at 500 °C. It has been found that the MFMOS (Metal-Ferroelectric-Metal-Oxide-Semiconductor) structure shows the least interface states density (less than 1011cm−2eV−1) with the maximal dielectric constant of PbTiO3 thin films.

The reaction of HMn(CO)5 with certain cyclopropenes when carried out in a detergent medium gives a different mixture of hydroformylated and hydrogenated products than is obtained when the same reaction is carried out in a homogeneous medium. These results are consistent with the intermediacy of caged geminate radical pairs whose escape from the cage is retarded by micelle sequestering.

The reaction of Ph3COH with 2 mol of HCo(C0)4 gives Ph3CH in quantitative yield. The reaction is cleanly second order (κ2  2.50 X 10-4 1 mol-1 s-1, in CH2Cl2 at 20°C), first order with respect to each reactant. The rate increases markedly with increase in solvent polarity, suggesting Ph3C+ as an intermediate. The rate of the reaction of HCo(C0)4 with is more than 103 as fast as with Ph3COH. No evidence for the functioning of HCo(C0)4 as a hydride donor could be secured.

Treatment of 3,3-diphenyl-1-propanol under cobalt-catalyzed hydroformylation conditions gives the corresponding aldehyde as the principal product but a small quantity of 1,1-diphenylethylene is also formed. Similarly 3-phenyl-1-propanol gives a small quantity of styrene. These arylethylenes are probably generated via the radical dehydrogenation to the corresponding aldehydes followed by decarbonylation and dehydrogenation, a reaction pathway that constitutes the reverse of the hydroformylation reaction. Attempts to rearrange isobutyraldehyde to the straight chain isomer (and the reverse) did not yield significant quantities of the rearranged product. The aldehydes obtained by cobalt-catalyzed hydroformylation apparently undergo the reverse reaction but at such a slow rate that the back reaction can usually be neglected.